Epithelial Wound Healing Enhanced by Transforming Growth Factor-α and Vaccinia Growth Factor
Gregory S. SchultzMichael J. V. WhiteRobert O. MitchellGregory L. BrownJenny LynchDaniel R. TwardzikGeorge J. Todaro
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Epidermal regeneration following middermal injuries to skin requires both proliferation and migration of keratinocytes. Epidermal growth factor (EGF) stimulates the proliferation of keratinocytes in culture, and topical administration of EGF accelerates epidermal regeneration of partial thickness burns or split-thickness incisions in vivo. Transforming growth factor-alpha (TGF-α) and vaccinia growth factor (VGF) have substantial sequence homology with EGF, and all appear to bind to the same receptor protein. Whether TGF-α or VGF can affect epidermal wound healing in vivo is not known. The present studies show that topical administration of TGF-α or VGF in antibiotic cream to partial thickness burns (second degree) accelerated epidermal regeneration in comparison with untreated or vehicle-treated burns. Low levels of both TGF-α and VGF (0.1 microgram per milliliter) appeared to be more effective than EGF in stimulating epidermal regeneration. Regenerated epithelium from burns treated with TGF-α or VGF appeared normal histologically. This finding suggests that topical application of selected growth factors may be useful in accelerating healing of partial thickness injuries.Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family of polypeptide growth factors, which includes EGF, transforming growth factor α (TGF-α), amphiregulin (AR) and betacellulin (BTC). To assess the potential role of HB-EGF in human gastric carcinomas, the expression of HB-EGF and EGF receptor (EGF-R) was examined in normal and cancerous gastric tissues and cultured gastric cancer cell lines. By Northern blot analysis, there was a 4.7-fold increase in HB-EGF mRNA levels in human gastric cancers compared with normal gastric tissues. There was a concomitant 3.9-fold increase in EGF-R mRNA levels in these cancers. Immunostaining revealed co-localization in 72% of the cancer cells of HB-EGF and EGF-R. AR and BTC moieties were not evident by Northern blot analysis. However, using PCR, both AR and BTC mRNA species were demonstrated in normal and cancerous gastric tissues. By Northern blot analysis, HB-EGF, TGF-α, AR, BTC and EGF-R mRNA moieties were co-expressed in KATO III and NCI-N87 gastric cancer cell lines. Furthermore, HB-EGF, EGF and TGF-α enhanced the growth of both cell lines in a dose-dependent manner. Our findings suggest that HB-EGF is relatively abundant in human gastric cancers and that co-expression of the EGF ligand family may lead to excessive activation of EGF-R in this disorder. © 1996 Wiley-Liss, Inc.
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Abstract The functional role of epidermal growth factor (EGF) in epithelium‐derived human colonic carcinoma cells was investigated by transfection with plasmid pUCDS3, which contained synthetic human EGF encoding sequences, into two human colonic carcinoma cell types with dissimilar phenotypic properties: the moderately differentiated and growth factor‐responsive Moser and the highly metastatic KM12SM cells. The Moser cells exhibited a proliferative response to treatment with exogenous EGF, while the KM12SM cells did not. The constitutive expression of the human EGF gene in these colonic carcinoma cell types resulted in elevated expression of EGF mRNA, with concurrent production and secretion of a large amount of EGF, and downmodulation of transforming growth factor‐alpha (TGF‐alpha) secretion. Growth stimulation and down‐modulation of both high and low affinity EGF receptors were observed in the EGF‐transfected Moser clones. Results of experiments using anti‐EGF and anti‐EGF‐receptor antibody to block the proliferation of EGF‐transfected Moser clones suggested that autocrine stimulatory mechanisms involving both EGF and TGF‐alpha were operative in these cells. By comparison, a growth‐inhibitory effect, with no apparent EGF receptor modulation, was observed in the EGF‐transfected KM12SM clones. Both the parental and EGF‐transfected KM12SM clones possessed fewer EGF receptors than the Moser cells, and anti‐EGF or anti‐EGF‐receptor antibody did not affect the cells' growth properties. These results suggested that the mechanisms of growth inhibition in the EGF‐transfected KM12SM clones were non‐autocrine or intracellular in nature. Thus, constitutive expression of the human EGF gene in two phenotypically different, epithelium‐derived human colonic carcinoma cells resulted in divergent altered growth characteristics.
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The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.
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It is widely accepted that polypeptide growth factors are involved in the growth and development of normal and neoplastic human prostate. It has been previously reported that epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) receptors are present in the human hyperplastic prostate tissue (BPH). To add information on the mechanism of action of EGF and transforming growth factor-alpha (TGF alpha), a peptide correlated to EGF, and the EGF receptor (EGF-R) in the human prostate, we studied the expression and cellular localization of messenger ribonucleic acid (RNA) encoding EGF, EGF-R, and TGF alpha in BPH tissue. Reverse transcriptase-PCR of total RNA extracted from BPH tissues documented the presence of specific transcripts for EGF, EGF-R, and TGF alpha. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that EGF, EGF-R, and TGF alpha messenger RNAs were mainly localized in the epithelial cells. Immunprecipitation and Western blot analysis showed that BPH tissue contained the corresponding proteins, EGF and TGF alpha. Our findings provide additional support for the idea that EGF and TGF alpha may be considered specialized symbols in the language of cell-cell interactions and for the hypothesis that in the human prostate they seem to act in an autocrine fashion.
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Human ectocervical epithelial cells are a primary target for infection by oncogenic papillomaviruses, which are strongly implicated as causative agents in the genesis of cervical cancer. Growth factors have been implicated as agents that stimulate proliferation and enhance the possibility of malignant transformation. In the present study we utilize several human papillomavirus (HPV) type 16-immortalized ectocervical epithelial cell lines to investigate the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on cell proliferation and the production of IGF binding proteins (IGFBPs). ECE16-1 cells, an HPV16-immortalized/nontumorigenic cell line, maintained in defined medium, produce and release high levels of IGFBP-3 (38/42 kDa) as well as smaller amounts of a 24-kDa IGFBP. Supplementation of defined medium with EGF causes a dose-dependent increase in cell growth and a concomitant decrease in the levels of IGFBP-3 released into the culture medium. EGF suppression of IGFBP-3 is maintained even when EGF-stimulated cell growth is suppressed 67% due to the simultaneous presence of 3 ng/ml of TGF beta 1, indicating that EGF suppression of IGFBP-3 levels is independent of EGF effects on cell growth. EGF suppression of IGFBP-3 production is correlated with a reduction in IGFBP-3 mRNA level. In the presence of EGF, the growth response of the cells to ng amounts of IGF-I is significantly enhanced. Moreover, the simultaneous presence of both EGF and IGF-I reduces the level of IGFBP-3 more efficiently than EGF alone. We also observe that the IGFBP-3 level is decreased and the 24-kDa IGFBP level is increased in HPV16-positive tumorigenic versus nontumorigenic cell lines. This is the first report of EGF acting as a positive regulator of IGF-I action via the IGFBPs. On the basis of these findings, we propose that EGF stimulates ECE16-1 cell growth via a dual-action mechanism by (a) stimulating growth directly via the EGF mitogenic pathway and (b) stimulating growth indirectly by reducing the levels of inhibitory IGFBPs and thereby potentiating the effects of IGF-I. In addition, the observation that more highly transformed cell types produce lower levels of IGFBP-3 and higher levels of 24-kDa IGFBP suggests that tumor cells in more advanced cervical cancers may have an altered response to IGF-I.
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The five members of the human epidermal growth factor (EGF) family (EGF, transforming growth factor alpha [TGF-alpha], heparin-binding EGF-like growth factor [HB-EGF], betacellulin, and amphiregulin [AR]) are synthesized as transmembrane proteins whose extracellular domains are proteolytically processed to release the biologically active mature growth factors. These factors all activate the EGF receptor, but in contrast to EGF and TGF-alpha, the mature forms of HB-EGF and AR are also glycosylated, heparin-binding proteins. We have constructed a series of mutants to examine the influence of the distinct precursor domains in the biosynthesis of AR. The transmembrane and cytoplasmic domains of the precursor are not required for secretion of bioactive AR from either COS or mammary epithelium-derived cells, although proteolytic removal of the N-terminal pro-region is less efficient in the absence of the membrane anchor. Deletion of the N-terminal pro-region, however, results in rapid intracellular degradation of the molecule with no detectable secretion of active growth factor. AR secretion is preserved by replacing the native pro-region with the corresponding domain of the HB-EGF precursor but not with that of the TGF-alpha precursor. In the absence of any N-terminal pro-region, secretion of the molecule is restored by deleting the N-terminal heparin-binding domain of mature AR. Both EGF and TGF-alpha, in contrast, can be secreted without their pro-regions. However, if the protein is fused with the AR heparin-binding domain, TGF-alpha secretion is inhibited unless the AR pro-region is also present. We propose that the heparin-binding domain of mature AR necessitates the presence of a specific structural motif in an N-terminal pro-region to permit proper folding, and thus secretion, of a bioactive molecule.
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양생 8일 부터 15일된 마우스 배자에서 TGF α 와 EGF의 분포 차이를 관찰하고자 실험을 시행하였다. 파라핀 포매에 의하여 만든 슬라이드를 TGF α 와 EGF 에 대한 항체를 사용하여 면역조직화학적 연구를 실시하였다. TGF α 의 엽색반용은 발생 제9일 마우스 배자에서 내배엽 중배엽 외배엽 기원의 여러 기관 즉 심장 눈술잔 머리중간엽 신경관 원시장관에서 관찰되었으며 발생 10일에서 15일된 배자에서는 그 염색 저도가 더 강하게 나타났다. EGF의 염색반응은 발생 제9일 마우스 배자에서는 중배엽 기원의 심장과 외배엽 기원의 원시장관에서 나타났으나 발생 10일에서 15일된 배자에서는 염색반응의 정도가 낮았다. 심장 발생에 있어 TGF α.는 EGF 보다 더 강력히 관계하였다. 폐 발생에 있어 TGF α의 염색반응은 기관지와 페싹 둘 다 에서 관찰된 반면 EGF는 단지 기관지에서만 염색반응이 나다났다. 신경계 발생에 있어 TGF α는 EGF 보다 더욱 광범위하고 강하게 나타났다. 골격계 발생에서 TGF α는 EGF 에 비하여 더욱 조기에 그리고 더 강하게 나다났다. 이런 결과는 TGF α와 EGF 가 마우스 발생 조절인자로 작용하며 특히 성장 신경계 중간엽 골격계 발생에서 TGF α가 EGF 보다 강하게 작용함을 추적할 수 있었다.
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